INTRODUCTION
It has been estimated that the annual number of snakebite accidents in the world is around 5 million, among which, 20,000 to 94,000 result in the death of the patient 1)(2)(3. In general, accidents caused by species within the Viperidae family (Bothrops spp., Crotalus spp.) are characterized by local damage to the tissues, including myonecrosis, dermonecrosis, hemorrhage, and edema 4. Myonecrosis is one of the most prominent effects induced by these venoms, causing the loss of tissue and permanent disability. This effect is produced mainly by the enzyme phospholipase A2 (PLA2; EC 3.1.1.4.), which hydrolyzes membrane phospholipids generating fatty acids which participate in the inflammatory process 5. The current main treatment for snakebite accidents is intravenous administration of equine immunoglobulins 6. However, the antivenoms available for bothropic accidents exhibit a limited neutralization of local and rapid installation effects 7.
Natural products are a rich source of pharmacologically active phytochemicals, which are beneficial as complex mixtures (single or mixed plant extracts), or as chemical backbones for new therapeutic agents. In fact, some plant extracts have been reported as antagonists of crude snake venoms and toxins 8)(9)(10)(11. Likewise, different types of plantderived compounds, such as flavonoids, coumarins, and other phenolic metabolites, have shown promise in neutralizing the PLA2 effects 12)(13)(14)(15. The species Swietenia macrophylla King (Meliaceae), also known as mahogany, was selected as a promising lead after screening thirty-six ethanolic leaf extracts, in a search for their neutralizing activity against Bothrops asper venom and PLA2 isolated from the venom of Crotalus durissus cumanensis16)(17. The tree is native to tropical America 18 and numerous biological properties have been reported for its leaves and twigs, including antidiabetic, tumor inhibition, anti-inflammatory, antifungal, antimalarial and insect antifeedant activities 19.
The purpose of this study was to identify the PLA2 inhibitors present in the crude extract of S. macrophylla that could be promising leads in neutralizing the local effects, and complement the traditional antivenoms. This involved the bioassay-guided fractionation of the ethanolic extract of the leaves of S. macrophylla, to characterize the metabolites that could be implicated in the observed bioactivity. Due to the challenges that snakebite accidents present in tropical countries, and the limited availability of antivenoms, the need for new alternatives to antagonize the activity of venoms and toxins is vital. In this way, the study of sustainable locally available species is highly relevant.
MATERIALS AND METHODS
Reagents
All solvents used for the extraction and partition procedures were of analytical grade (Merck, Darmstadt, Germany). Reagents for the phytochemical analysis and the (+)-catechin (98% purity) used for bioassays, were from Sigma-Aldrich® (St. Louis, MO, USA). The derivatizing agent used (BSTFA+TMCS) was purchased from Supelco (Bellefonte, PA, USA).
Plant material
For the extraction and bioassay-guided fractionation, S. macrophylla leaves (2 kg) were collected in the tropical and pre-mountain forests of Medellin, Colombia, at 1600 m.s.l (6º15'41” N, 75º34'35.5” W). The voucher specimen, labeled as TL-103, was deposited in the Herbarium of the Universidad Nacional, Gabriel Gutiérrez Villegas (MEDEL) and identified by Professor Mauricio Sanchez Saenz (Universidad Nacional de Colombia, sede Medellin).
Extraction and bioassay-guided fractionation of Swietenia macrophylla
The leaf plant material was dried at room temperature, milled, and extracted by percolation at room temperature overnight with 90% ethanol (1L x 100 g). The ethanol extract was filtered and concentrated under reduced pressure, using a rotary evaporator (Büchi R-144) at a temperature below 40°C. The resultant ethanolic extract was mixed with distilled water to 10%, and then defatted with hexane. Subsequently, the ethanolic aqueous extract was evaporated and lyophilized.
For the fractionation of the resulting S. macrophylla extract (140 g), silica gel 60 F254 open column chromatography was carried out, using a gradient of dichloromethane-acetone-methanol, beginning with the less polar solvent and ending with methanol. According to the chromatographic profiles obtained by thin layer chromatography (TLC), twenty-five initial fractions were combined to afford eight final fractions that were evaluated in the bioassays described below.
Characterization of compounds using gas chromatography coupled with mass spectrometry (GC-MS)
The chemical profiles of the ethanolic extract and the most promising biologically active fractions of S. macrophylla were analyzed using a gas chromatograph (Agilent 6890), coupled with a mass spectrometer (Agilent 5973), employing a capillary column of fused silica (Agilent HP-5, 0.25 mm x 30 m x 0.25 μm) covered with 5% phenyl methyl siloxane. All of the samples (extract and fractions) were derivatized before injection according to the method described by Silici and Kutluca 20. One mg of each sample was diluted in 50 μL of pyridine and a mixture of 100 μL of BSTFA (N,O-bis( trimethylsilyl) trifluoroacetamide) with 1% of TMCS (trimethylchlorosilane) was added. The mixture was heated for 30 min at 100ºC. For each sample, 5.0 μL was injected, using helium gas grade 5 (AGA Fano S.A., UAP 99.999 %) at a flux of 1.0 mL/min (linear velocity 37 cm/s). The injection used the split-less mode with an initial temperature of 200ºC for 3 min, which was raised to 250ºC and maintained for 1 min. Finally, the temperature was raised by 2ºC per minute to a maximum of 350ºC for 60 min. To obtain the mass data, the detector was fixed at 350ºC. The run was made using the SCAN mode between m/z 30-800.
The chromatograms were analyzed with AMDIS software (Automated Mass Spectral Deconvolution and Identification System), and the spectral database NIST 98 (2001). The identification of compounds was done through comparison of the mass spectral fragmentation patterns of each compound with the databases mentioned.
Determination of (+)-catechin in Swietenia macrophylla
The confirmation and quantification of (+)-catechin in the ethanolic extract and active fractions of S. macrophylla was performed through HPLC analysis. The separation of the compound was done using an ultra-aqueous C18 column with a particle size of 5 μm (250 mm x 4.6 mm, Merck). As a mobile phase, methanol (A), and formic acid (0.1%) were used in a gradient system of elution of 0.01 min 60% of A; 5-12 min 80% of A; 13-14 min 60% of A. The mobile phase flux was 1.0 mL/min. The identification was done by comparison with a standard of (+)-catechin (Sigma).
Venoms and toxins
Crotalus durissus cumanensis (Colombian rattlesnake), and Bothrops atrox venom was obtained from specimens from Meta (southeast region of Colombia) and B. asper venom was obtain from specimens from Antioquia and Chocó (western region of Colombia). Venoms were obtained by manual extraction of the specimens that are kept in captivity at the Serpentarium (Universidad de Antioquia, Colombia). Venoms were centrifuged at 800 g for 15 min, and supernatants were lyophilized and stored at -20 ºC until used.
Purification of PLA2s
The Lys 49 PLA2 was obtained from B. atrox, and Asp 49 from C. d. cumanensis. Each venom was initially chromatographed by cationic interchange on CM Sephadex and molecular exclusion on Sephadex G-75 respectively. Peak of interest were purified by reverse-phase HPLC on C18 column eluted at 1 mL/min with a gradient from 0% to 100% acetonitrile in 0.1% trifluoroacetic acid (v/v). Absorbance in the effluent solution was recorded at 280 nm 21. Snake venom PLA2 are enzymes that are able to resist extreme chemical conditions, such as low pH, higher ion concentrations and temperature, among others 21)(22. In addition, several studies have demonstrated that snake venom PLA2 can be purified by HPLC using acetonitrile without changes in their enzyme and biological activities 21)(23)(24.
Inhibitory activity of PLA2 using egg yolk as substrate
The inhibitory activity of the extract, fractions and compounds against the PLA2 present in the venom of B. asper was assayed using egg yolk phospholipids suspended in 1% Triton X-100, 0.1 M Tris HCl, 0.01 M CaCl2, buffer (pH 8.5) according to the method reported by Dole 25. Measurement of the inhibition of the PLA2 activity was conducted at different concentrations (w/w toxin:fraction) with 10 μg of venom in the presence of the substrate. Samples were incubated for 30 min at 37°C before calculation of the inhibition percentage, where 0 % is the activity induced by PLA2 alone.
Inhibitory activity of PLA2 using 4- Nitro- 3-octanoyloxy-benzoic acid (4N3OBA) as substrate
The measurements of enzymatic activity using the monodispersed substrate 4N3OBA were performed according to the method described by Holzer and Mackessy 26, and adapted for a 96-well ELISA plate. The Asp 49 PLA2 isolated from C. d. cumanensis was used in this assay. The standard assay contained 200 μL of buffer (10 mM Tris-HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0), 20 μL of 10 mM of substrate (4NO3BA), 20 μL of sample (20 μg PLA2 or 20 μg PLA2 + several concentrations of active fraction or (+)-catechin) and 20 μL of water. The negative control was only the buffer solution. The inhibitory effect of the active fraction F5 and (+)-catechin on PLA2 activity was determined through co-incubation of the enzyme with each concentration of the compound for 30 min at 37°C. After the incubation period, the sample was added to the assay and the reaction was monitored at 425 nm for 40 min (at 10 min intervals) at 37°C. The quantity of released 3-hydroxy-4-nitrobenzoic acid was proportional to the enzymatic activity, and the initial velocity (Vo) was calculated considering the absorbance measured at 20 min.
Inhibition of cytotoxic activity induced by PLA2
The inhibitory ability of crude extract and fraction F5 on the cytotoxicity caused by B. asper venom or a Lys 49 PLA2 isolated from B. atrox venom were evaluated using murine skeletal muscle C2C12 myoblasts (ATCC CRL-1772), as reported 27. For the assay, 100 μg of toxin or 20μg of B. asper were incubated for 30 min at 37°C with S. macrophylla or their F5 fraction and diluted in 150 μL of medium DME (Dulbecco's Modified Eagle's Medium supplemented with 1% fetal calf serum). After this period, the mixture was added to the plates (150 μL/plate) and incubated for 3 h at 37°C. An aliquot of the supernatant was collected to detect the activity of the enzyme lactate dehydrogenase (LDH; EC 1.1.1.27) through a kinetic method (Wiener LDH-P UV). Reference controls to determine the 0 and 100 % of cytotoxicity were the medium and the medium with 0.1% (v/v) of Triton X-100, respectively. Additional controls consisted of incubated cells with samples and without the toxins. The results are expressed as inhibition percentages, considering the toxin and the culture media as 100 and 0 % of activity, respectively.
Molecular docking studies using (+)-catechin as ligand
The program Avogadro 1.1.0 28 was used to build the (+)-catechin molecule showed in Figure 1. The same software was used to improve its overall structure using an energy minimization process based on the MMF94 force field by means of a steepest-descent algorithm in 500 steps. Molecular docking was carried out on a personal computer using Autodock Vina 29. The PLA2 (PDB code 2QOG) from C. d. terrificus showed 57% homology in the N-terminal with the Asp49 PLA2 used in these studies 21. Protein was used with the exclusion of water molecules. The structure of the protein was prepared using the Protein Preparation module implemented in the Maestro program. First, hydrogen atoms were automatically added to each protein according to the chemical nature of each amino acid, on the basis of the ionized form expected in physiological conditions. This module also controls the atomic charges assignment. Second, each 3D structure of the protein was relaxed through constrained local minimization using the OPLS (Optimized Potentials for Liquid Simulations) force fields in order to remove possible structural mismatches due to the automatic procedure employed to add the hydrogen atoms. When necessary, bonds, bond orders, hybridizations, and hydrogen atoms were added, charges were assigned (a formal charge of +2 for the Calcium ion) and flexible torsions of ligands were detected. The α-carbon of His48 was used as the center of the grid (X = 44.981, Y = 27.889 and Z = 46.392), whose size was 24 Å3. Exhaustiveness = 20. Then, the ligand poses with the best affinity were chosen, and a visual inspection of the interactions at the active site was performed and recorded. Molegro Molecular Viewer (MMV 2.5.0, http://www.clcbio.com/products/molegro/#molecular-viewer ) and UCSF Chimera (www.cgl.ucsf.edu/chimera/) were used to generate docking images.
Statistical analysis
To identify the variation in the biological activity, a one-way analysis of variance (ANOVA) was carried out. When significant differences were detected (α ≤ 0.05), a Tukey's range test, with a confidence level of 95%, was done to establish the differences in each level of activity. All tests were done in triplicate and are expressed as the mean ± standard deviation using R, version 2.15 30.
RESULTS
Biological activity of extract and fractions
The ethanolic extract of S. macrophylla leaves exhibited an 87.2 ± 11.0% effect in neutralizing the phospholipase enzymatic activity of the B. asper venom using egg yolk as substrate. Furthermore, as mentioned Table 1, there was a differential in the activity between the eight fractions derived from the leaf extract, with the most significant activity found in fraction F5.
Because of the promising results obtained with fraction F5, it was selected for further study. As described Table 2, Fraction F5 and (+)-Catechin inhibited the PLA2 activity of B. asper venom, in a dose-dependent way. Also the ethanolic extract and fraction F5 showed inhibition of the cytotoxicity induced by the B. atrox venom and their Lys 49 PLA2 (80 and 100% respectively).
*Results expressed as the mean ± standard deviation (n=3)
**The different letters represent significant differences among the results.
Characterization of fraction F5 of Swietenia macrophylla
In the mass spectra of the ethanolic extract and fraction F5 of S. macrophylla the fragmentation patterns of phenolic compounds were seen. Catechin was detected at the end of the elution period as shown in Figure 2B. A compilation of the proposed identifications of the compounds found in the extract and fraction F5 through mass spectral analysis are shown in Table 3. Through HPLC, it was possible to confirm that the isomer of catechin in fraction F5 was (+)-catechin, at a concentration of 0.184 mg/mL.
Bioactivity of (+)-Catechin
Due to the high concentration of (+)-catechin in fraction F5, additional biological evaluation was carried out on the pure compound. As described Table 2 , (+)-catechin inhibited the PLA2 enzymatic activity of the B. asper venom, in a dose dependent way. Moreover, to support the hypothesis that the biological activity observed for fraction F5 is mainly due to the presence of (+)-catechin, a spectrophotometric assay was done to quantify the inhibition on the enzymatic activity of Asp49 PLA2 purified from the venom of C. d. cumanensis. As substrate, 4N3OBA was used and 18% of (+)-catechin, in an attempt to reflect the same percentage of this compound present in fraction F5, as determined by the GC-MS analyses. In this case, the inhibition of the PLA2 by (+)-catechin was 83.1 ± 3.1%. In addition, (+)-catechin inhibited by 83.3 ± 11.2% the liberation of lactate dehydrogenase induced by Lys 49 PLA2 from B. atrox in cytotoxicity assay.
Molecular docking
To support the results obtained with the bioassays of (+)-catechin, and in an attempt to explain the mechanism of neutralization of the PLA2 by this metabolite, molecular docking studies of (+)-catechin were performed. The observed binding free energy of (+)-catechin was -8.6 kcal/mol. Docked solution with the lowest binding energy was selected and described. Docking results are displayed in Figure 3 an they suggested that OH from 4´ and 5' carbons of (+)-catechin could form hydrogen bonds with carboxylate moiety of residue Asp49, while OH from 5 could form a hydrogen bond with Asn6 (Figure 2). In addition, our results suggested a π-π stacking interaction between rings A of (+)-catechin with that of the residues Phe5 and Tyr22 of the toxin; and ring B of the flavonoid and Tyr52 of the enzyme. Additional Van der Waals interactions with Ala18, Ala23, Cys29, Gly30, His48 and Lys69 were also detected (distance lower than 3.5 Å). As shown in Figure 4, the effect of (+)-catechin in blocking the hydrophobic channel of the enzyme may be observed.
DISCUSSION
In this study, the inhibitory activity of 87.2 ± 11.0% for the extract led to bioassay-guided fractionation, wherein all the fractions exhibited activity, but the most significant inhibitory potential was observed for fractions F4 and F5. Fraction F5 was also evaluated against PLA2s present in B. asper venom, and dose-dependent activity was exhibited. These results are comparable to those reported by Pereañez et al. 16, in which an inhibition level of 59.3 ± 3.5% was found for the crude extract of the leaves of S. macrophylla. Similarly, the total inhibition of the cytotoxic activity caused by a Lys49 PLA2, by fraction F5 was evidenced. Pereañez et al. 17 had reported inhibition of the edema induction, myotoxic and anticoagulant activities induced by an Asp49 PLA2, from C. d. cumanensis venom. Moreover, given the promising bioactivity of fraction F5, in the characterization of the major compounds by GC-MS it was relevant to find two phenolic compounds in the derivatized fraction F5, namely protocatechuic acid, and catechin. Protocatechuic acid is an antioxidant that also has inhibitory capacity against the PLA2 of snake venoms 31. Likewise, various isomers of catechin have been studied because of their inhibitory activity of PLA2 present in snake venoms 12)(32. The presence of these two compounds was proposed based on their mass spectral fragmentation patterns, and confirmed by comparison with the spectral database NIST 98. The data obtained with GC-MS for these compounds was in accordance with the literature 33. In this way, the TMS derivative proposed as protocatechuic acid presented a molecular ion with m/z 370 and a base peak at m/z 193. For the flavan- 3-ol, catechin, the derivatized sample exhibited a molecular ion with m/z 650 and a characteristic base peak of m/z 368 from the excision of the heterocyclic ring through a Retro-Diels Alder fragmentation were the keys to identify this compound 34.
The fact that fraction F5 had the most promising activity, and since catechin was a major compound within this fraction (Table 3), led to a study of the activity of catechin against PLA2. For the biological activity, a commercial sample of (+)-catechin was used, and the determination of the isomer and its purity was made through HPLC. As observed in Table 3, (+)-catechin was a promising compound with dose-dependent activity. Additionally, in the spectrophotometric assay using the monodispersed substrate 4N3OBA, (+)-catechin presented inhibition of PLA2. For this assay, the same concentration of (+)-catechin present in the fraction F5 was used to keep a ratio of 1:10. In this way, it was suggested that the percent inhibition of the enzyme PLA2 by (+)-catechin is similar to that in the fractions, using the same ratio venom: compound. This was done to determine if (+)-catechin was the main compound responsible for the bioactivity of fraction F5, which can be positively suggested based on the results obtained and displayed in Table 2.
Another significant finding was the total inhibition of the cytotoxicity induced by Lys49 PLA2s from B. atrox, observed by fraction F5 suggesting that it reduces the myonecrosis induced by this type of toxins. The Lys49 PLA2 in relation with Asp49 showed several substitutions in amino acids residues, but the replacement of amino acid Asp49 by the amino acid Lys, is the principal cause that leads to a loss of the enzyme activity, especially because it damages the loop that binds to calcium 35)(36. Nevertheless, it is reported that the Lys49 PLA2 induces local myotoxicity and edema through a mechanism that is not well established, in which a participation of the C-terminal region has been proposed 35. In this way, to find a compound that inhibits this kind of toxins is a relevant outcome. The polyphenol rosmarinic acid isolated from diverse species of the Boraginaceae and Lamiaceae, reduced 80% and 90 % the muscle damage and the neuromuscular blockade induced by PrTX-I (a PLA2 Lys 49 from the venom of Bothrops pirajai) on mice neuromuscular (phrenic-diaphragm) preparations. X-ray crystallographic studies between two molecules demonstrated that rosmarinic acid obstruct the entrance of the hydrophobic channel in PrTX-I affecting the interaction with membrane 37.
The molecular docking experiment performed with an Asp49 PLA2 enlightens how (+)-catechin can bind to the PLA2 active site. In this way, this metabolite can interact by hydrogen bonds mediated by the hydroxyl at carbons-4´and 5´of ring B and the hydroxyl of carbon-3 of ring C with Asp49, which, in this case, is one of the residues implicated in the coordination of calcium required for the enzymatic catalysis. As a result, this interaction of (+)-catechinenzyme could destabilize the cofactor binding. In the same way, it is evident that (+)-catechin could form Van der Waals interactions with residues on the interfacial binding surface (Asn6, Ala 18, and Lys69), which is the region of the PLA2 that allows adsorption of the enzyme onto the lipid-water interface of the phospholipids membrane bilayer. Thus, these interactions could block the recognition of aggregated phospholipids. All of the interactions mentioned, suggest the mode of interaction of (+)-catechin with PLA2s. Lindahl and Tagesson 38 informed that hydroxyl groups in 3´and 4´-position in the B-ring, 5-hydroxyl group in the A-ring, unsaturation and the 4-oxy in the C-ring appear to be important for the overall ability of flavonoids to inhibit PLA2 activity. (+)-Catechin has some of these structural characteristics, which could support the inhibitory activity of the compound. In addition, it is important to mention that these authors also stablished that removal or addition of one hydroxyl group in the B-ring retains a strong PLA2 inhibition but leads to a decreased selectivity towards PLA2 from group II 38. This information is in agreement to that reported by Lättig and collaborators 39, which points out the significance of hydroxyl groups in flavonoids for the inhibition of PLA2 type II.
CONCLUSIONS
The crude extract of the leaves of Swietenia macrophylla exhibited strong inhibitory activity against the PLA2s enzymes present in the venoms of B. asper, B atrox and C. d. cumanensis,. Catechin, is one of the components of the most active fraction, F5. In molecular docking studies, (+)-catechin was shown to bind to the active site, and interacts through hydrogen bonds with the amino acid Asp49, which is implicated in the calcium chelation (enzyme cofactor). It is also suggested that (+)-catechin blocks the recognition of aggregated phospholipids, while the aromatic rings A and B can bind through π-π interactions to the aromatic rings of residues Tyr22, Phe5, and Tyr52.
ACKNOWLEDGMENTS
Authors are thankful to Professor Emeritus Geoffrey Cordell, University of Florida, for his suggestions and for improving the text. L. Preciado acknowledges the School of Chemistry for teaching assistantships during the period of her graduate student training. The authors thank the Comité para el Desarrollo de la Investigación and Sostenibilidad 2015-2016 and Project CIQF-217, Universidad de Antioquia and Universidad Nacional Colombia, sede Medellín.